This invention relates to capillary electrophoresis.
One class of capillary electrophoresis system includes as its principal parts: (1) a length of capillary tube filled with an appropriate buffer; (2) a sample injector for injecting a small quantity such as 1 to 30 nl (nanoliters) of sample mixture into one end of the length of capillary tube; (3) means for applying an electrical potential between the two ends of the length of capillary tube which potential is normally applied by a high-voltage, low-current power supply; and (4) an on-line detector. An example of this type of equipment is described in U.S. application Ser. No. 07/277,566 filed Nov. 29, 1988, in the name of Robert William Allington and assigned to the same assignee as this application, the disclosure of which is incorporated herein by reference.
In use, the capillary tube first is filled with an appropriate buffer solution or gel and then a small volume of sample introduced into one end of the capillary. Next the ends of the capillary tubing are inserted into two separate vessels each containing: (1) a predetermined volume of the same buffer solution that is in the capillary tube; and (2) a platinum electrode. The electrodes are connected to the power supply terminal cables, after which the power supply is turned on. The separated components or bands are detected by the detector.
In one type of prior art detector used in one class of capillary electrophoresis system, light is transmitted between the light source and the light sensor through the entire side of the capillary tube with light blocking members so as to utilize maximum light flux.
These two types of detectors each have a disadvantage. Without at least one single narrow aperture, some of the light flux passes through the medium to the detector, but other parts of the light flux bypass the medium. In such an embodiment, the flux passes around the medium through the walls of the quartz capillary tube and to the detector. This bypassed light results in a high level of background light which reduces the sensitivity of the detector.
A conventional method of reducing background light is to use one narrow slit and collimated light. This arrangement, if applied to capillary electrophoresis, has a disadvantage in that the light level at the detector is so low that there is excessive noise in relation to the strength of the electrical signals generated by the detector. This excessive noise is quantum noise.
One type of sample injector described in U.S. patent application Ser. No. 07/277,566 filed Nov. 29, 1988, injects very small samples by applying a vacuum on the end of the separating apparatus opposite to the source of the sample to draw a small portion of the sample into the separating apparatus.
This type of sample injector controls the amount of sample by timing the period through which the vacuum is applied. The time is determined by experience. This type of sample injector has a disadvantage of being bulky and expensive if it is to be precise.
Electromigratory sample introduction is also known. The general technique of electromigration sample injection for capillary electrophoresis is described by James W. Jorgensen in "New Directions in Electrophoretic Methods", ACS Symposium Series 335, American Chemical Society Washington, D.C., page 185 (September, 1985), the text of which is incorporated herein by reference. Electromigration or electrokinetic sample introduction involves placing both capillary tube inlet and high voltage electrode in the sample vessel and briefly turning on the power supply. A small amount of sample is drawn into the capillary by electrophoresis or electroosmotic flow which is caused by high voltage applied across the capillary ends. Then the power supply is turned off, the capillary and electrode are repositioned in the buffer reservoir, and the power supply is then turned on again to perform the separation.
Electromigratory sample introduction has several disadvantages, such as for example: (1) it introduces polar molecules at rates dependent on their charge and thus creates uncertainty; (2) some molecules may have a polarity that prevents their introduction; and (3) it can be expensive and difficult to use.
Split flow sample injectors are known in gas chromatographic systems and some liquid chromatographic systems. For example, the use of split injection is described in "Pressure and Composition Gradients in Capillary Supercritical Fluid Chromatography" by Clement R. Yonker and Richard S. Smith, Anal. Chem., Mar. 1, 1989, 59, 727-731. Moreover, M. Gene et al. describes a sampler in "Electric Sample Splitter for Capillary Zone Electrophoresis", J. Chromatography 320, 159-165 (1985). This sampler works on the principle of secondary electrophoresis rather than split flow injection. It has the disadvantage of requiring a second power supply for the second electrophoresis direction.
The prior art systems, however, have several disadvantages such as: (1) they are used with sample injectors or high pressure flow apparatus and are not adapted for low velocity controlled manual injection such as that available by syringe injection; (2) it is difficult to isolate 30 kilovolts across the different "valve" positions normally used, which are the "load sample/inject" and "run" positions; and (3) there is substantial carryover from one run to the next. The need for complications such as a second power supply are undesirable features of some prior art systems.